Human melanocyte transformation

Our long-term objective is to understand the mechanisms for transformation of normal human melanocytes to malignant melanoma expecting that this work will yield the identification of new targets for therapy. The presence of BRAF mutations in the majority of non-malignant nevi suggests that this gene is necessary but not sufficient for inducing melanoma. In humans, we do not know which genes are required to cooperate with BRAF. When BRAFV600E is over-expressed in normal human melanocytes, cells undergo senescence suggesting that survival mechanisms need to be established before additional genetic abnormalities can occur. Our working hypothesis is that host-derived growth factors and cytokines produced by ultraviolet (UV)-activated keratinocytes and fibroblasts allow survival of mutant BRAF-expressing melanocytes/nevus cells whereas UV and UV-induced inflammatory mediators damage melanocytes and/or eliminate immune surveillance. To account for the unique position of human melanocytes at the basement membrane of the epidermis, our laboratory has developed skin reconstructs (also called skin equivalents or synthetic skin) consisting of a "dermis" with fibroblasts embedded in a collagen or fibrin matrix and melanocytes and keratinocytes in the "epidermis", where the keratinocytes form multiple layers once they are exposed to air. For long-term experiments including UV irradiation, such skin reconstructs can be grafted to immunodeficient mice where they become an integral part of the murine skin throughout the lifetime of the animals. In the first aim, we will determine the role of fibroblasts and keratinocytes in BRAF-mediated melanocyte transformation and test the hypothesis that growth factors and cytokines secreted by UV-activated fibroblasts and/or keratinocytes help melanocytes survive and overcome oncogene (BRAF V600E)-induced senescence. We will test our hypothesis in human skin reconstructs maintained in vitro and in vivo. In the second aim we will determine the role of UV-induced human inflammatory mediators for melanocyte transformation to melanoma by reconstituting mice, which lack murine B-, T-, and NK-cells, with human CD34 + hematopoietic stem and progenitor cells, graft them with human skin reconstructs containing melanocytes, and irradiate the grafts with UV. These studies are expected to demonstrate that growth factors from the skin microenvironment ensure survival/growth of melanocytic cells whereas UV and the resulting inflammation induce DNA damage or down- modulate the immune system.